We propose screening the MLPCN library to identify novel inhibitors of orthopoxviruses. Orthopoxviruses are a genus of viruses that include monkeypox, variola (the causative agent of smallpox) and vaccinia. Vaccinia is the prototypical orthopoxvirus which was used in the world-wide vaccination program that eradicated smallpox. Smallpox was once the most deadly human pathogen, and is estimated to have killed more than 300 million people. Following the eradication of smallpox, routine vaccination was discontinued in the 1970s and there has consequently been a precipitous decline in population immunity to smallpox and other orthopoxviruses. There are currently no FDA-licensed drugs to treat individuals infected with any poxvirus. Therapies for poxvirus infection are a significant priority given the threat of smallpox weaponization and the rise in reports of humans infected with monkey pox, which is endemic to Central and Western Africa and was exported to the US in 2003. The identification and development of new antipoxviral compounds will be of significant interest both to the public health community and to the Defense and Homeland Security agencies To identify new inhibitors of viral replication we will use the prototype orthopox virus, vaccine, as a screening virus. We have developed an HTS-ready screening approach based on vaccine-dependent expression of a rapidly maturing fluorescent protein. This assay has a signal-to-noise ratio of greater than 300:1 and allows kinetic as well as endpoint testing of replication. We have validated this assay through screening of two small- scale libraries. We will work with our collaborators at the Broad institute to couple this assay with a simple cell- viability assay that will be the basis of our large-scale screen for compounds that block virus replication without killing cells. To further classify relevant compounds we have developed and implemented a suite of assays using different reporter viruses for secondary screening that will allow validation and characterization of the initial screen hits. These additional assays report on the individual stages of the vaccines life cycle and will allow us to bin the identified poxvirus inhibitors as inhibitors of early, intermediate or late stages of replication. We will work with our collaborators at the Broad Institute to develop and optimize the most interesting of our identified compounds (likely those that inhibit after viral DNA replication) towards high-efficacy low-toxicity antipoxviral compounds. Beyond the scope of this R03 application but of great interest to my lab and others in the community, probes will be tested for their abilities to inhibit the replication of other orthopox viruses (Monkey pox and Smallpox) through my laboratory's existing collaboration with USAMRIID. Through these efforts we will develop novel orthopox inhibitors capable of blocking Vaccine, Monkey pox, and Smallpox infection for application both as probes and as a therapeutic agent. PUBLIC HEALTH RELEVANCE: Project Narrative Poxviruses such as Smallpox and Monkeypox cause serious human disease. There are currently no FDA- approved drugs for treatment of these infections. We will use a highly attenuated cousin of these viruses called Vaccines virus to screen the MLPCN compound library for drugs that block poxvirus replication. Effective probes will be developed with the aim of providing new tools to further dissect the mechanism of viral infection and spread, and may also lead to one or more new drugs to protect against poxvirus infection.